ABSTRACT

Context Individuals with elevated levels of homocysteine tend
to have higher prevalence of cardiovascular disease. However,
prospective studies of homocysteine are inconsistent and data among
women are limited.

Participants From a total cohort of 28,263 postmenopausal
women with no history of cardiovascular disease or cancer at baseline,
122 women who subsequently experienced cardiovascular events were
defined as cases, and 244 age- and smoking status–matched women who
remained free of disease during follow-up were defined as controls.

Results Of the 122 cases, there were 85 events of MI or stroke and
37 coronary revascularizations. Case subjects had significantly higher
baseline homocysteine levels than controls (14.1 vs 12.4 µmol/L;
P=.02). Subjects with homocysteine levels in
the highest quartile had a 2-fold increase in risk of any
cardiovascular event (relative risk [RR], 2.0; 95% confidence
interval [CI], 1.1-3.8). This effect was largely due to an excess of
cases with high levels of homocysteine; the RR for those with
homocysteine levels at or higher than the 95th percentile (20.7
µmol/L) was 2.6 (95% CI, 1.1-5.7). Risk estimates were independent
of traditional risk factors and were greatest for the end points of MI
and stroke (RR for those with baseline homocysteine levels in the top
quartile, 2.2; 95% CI, 1.1-4.6). Self-reported multivitamin supplement
use at study entry was associated with significantly reduced levels of
homocysteine (P<.001). However, the association between
increasing quartile of homocysteine level and risk of MI or stroke
remained significant in analyses controlling for baseline multivitamin
supplement use (P=.003 for trend), and
subgroup analyses limited to women who were
(P=.02 for trend) or were not
(P=.04 for trend) taking multivitamin
supplements.

Several mechanisms
have been proposed linking hyperhomocystinemia to vascular damage, and
it has been hypothesized that elevated levels of total plasma
homocysteine represent an important modifiable risk factor for
atherothrombotic disease.1,2 A number of retrospective and
cross-sectional studies provide support for this
hypothesis.3 However, because homocysteine levels may
increase following acute myocardial infarction (MI)4 or
stroke,5 any observed association could, at least in
theory, be a result rather than a cause of acute vascular occlusion.

In contrast, in prospective studies, homocysteine levels are
ascertained prior to the onset of thrombosis and, thus, reduce the
potential for this type of bias. The results of such studies, however,
have been inconsistent. For example, prospective studies from the
United States6 and Finland7 report no evidence
of association between baseline homocysteine level and subsequent
coronary heart disease risk, whereas another study of middle-aged US
men reported a positive association in the subgroup in the top 5% of
the homocysteine level distribution8 that was no longer
present with long-term follow-up.9 In this latter study,
there were no associations between homocysteine and risk of
thromboembolic stroke10 or incident angina
pectoris.11 Similarly, among men enrolled in
the prospective Atherosclerosis Risk in Communities (ARIC) study, no
association was found between homocysteine levels and subsequent
coronary events.12

On the other hand, prospective studies from Norway13,14 and
Great
Britain15 demonstrate positive
associations with coronary heart disease, while other studies suggest
positive associations for peripheral arterial disease16 and
stroke.17 Furthermore, among women enrolled in the ARIC
study, a positive univariate association was reported; however, after
adjustment for other coronary risk factors, this effect was no longer
statistically significant.12 Thus, recent prospective data
are less consistent than prior retrospective and cross-sectional data.

To provide further prospective data in women, we evaluated baseline
levels of homocysteine among apparently healthy participants in the
Women's Health Study (WHS), an ongoing primary prevention trial among
39,876 postmenopausal women with no history of cardiovascular
disease or cancer,18 and related these levels to the future
risk of experiencing cardiovascular events.

METHODS

We used a prospective, nested case-control study design among
participants in the WHS, an ongoing, randomized, double-blind,
placebo-controlled trial of aspirin (100 mg on alternate days) and
vitamin E (600 IU on alternate days) in the primary prevention of
cardiovascular disease and cancer. Cohort assembly for the WHS occurred
between 1993 and 1995. Baseline blood samples were obtained in EDTA
from 28,263 (71%) of WHS study participants and stored in
liquid nitrogen until analysis.

In this study, cases were defined as WHS participants
who provided an adequate baseline blood sample and subsequently
experienced a cardiovascular event (defined as death due to
coronary heart disease, nonfatal MI or stroke, percutaneous
transluminal coronary angioplasty, or coronary artery bypass graft)
during a 3-year follow-up period. The diagnosis of MI was made if
symptoms met World Health Organization criteria and the event was
associated with cardiac enzyme abnormalities or diagnostic
electrocardiogram changes. The diagnosis of stroke was made if the
patient had a new neurologic deficit lasting more than 24 hours;
computed tomography or magnetic resonance imaging scans were available
in the majority of cases. Reported percutaneous transluminal coronary
angioplasty and coronary artery bypass graft procedures were confirmed
by hospital records. Coronary heart disease deaths were confirmed by
autopsy reports, death certificates, and circumstances of death. All
end points were adjudicated by a committee of cardiologists and
neurologists.

For each case subject who had a confirmed cardiovascular event during
follow-up, 2 control subjects of the same age (±1 year) and
smoking status (past, current, or never) were selected from the
remaining WHS participants who also provided baseline blood samples and
who reportedly remained free of cardiovascular disease during the
3-year follow-up. Using these matching criteria, 122 cases and 244
controls were selected and form the basis for these analyses.

Baseline plasma samples from each case and control subject were thawed
and assayed for total plasma homocysteine level using the IMx
homocysteine assay (Abbott Laboratories, Abbott Park,
Ill).19,20 The assay coefficient of variation ranges from
3.7% to 5.3% when measuring samples between 5 and 25 µmol/L. All
investigators and laboratory personnel were blinded to the subjects'
clinical details. Case and control specimens were handled identically
and in blinded fashion throughout the blood collection, storage,
retrieval, and analysis process. Samples were analyzed in triplets,
with the position of the cases varied at random within triplets to
avoid systematic bias and interassay variability.

Means and proportions for baseline cardiovascular risk factors were
calculated for cases and controls. The significance of differences in
means between the case and control groups was tested using the
t test, while the significance of differences in proportions
was tested using the χ2 statistic. Tests for trends were
used to assess for evidence of a linear relationship between increasing
level of homocysteine and the risk of future coronary events
after dividing the sample into quartiles, defined
by the distribution of the control values. Adjusted estimates of risk
were obtained using logistic regression models that, in addition to
accounting for the matching variables of age and smoking status,
controlled for randomized treatment assignment, history of
hyperlipidemia, history of hypertension, body mass index (weight in
kilograms divided by the square of height in meters), exercise
frequency (per week), history of diabetes, and parental history of MI
before age 60 years. To compare our results with those of previous
investigations, we further computed from these linear models the
relative risk (RR) associated with each 5-µmol/L increase in total
plasma homocysteine. In addition, because some prior studies have
suggested that a threshold effect exists for homocysteine, the data
were also analyzed using prespecified cutoffs at the 50th, 75th, 90th,
and 95th percentiles of homocysteine level among the control values.
Additional analyses were performed after adjustment for baseline
multivitamin supplement intake and in subgroups limited to women who
did and did not report multivitamin supplement use at study entry. All
P values are 2-tailed and all confidence intervals (CIs) were
calculated at the 95% level.

RESULTS

Due to matching, the distributions of age and smoking status were
virtually identical in the case and control groups. As expected in a
study of cardiovascular disease, cases were more likely to have a
history of hyperlipidemia, obesity, hypertension, or diabetes or a
family history of premature MI. There were no significant differences
for self-reported levels of alcohol consumption, exercise frequency,
hormone replacement therapy, or multivitamin supplement use
at baseline (Table 1).

Of the 122 cases, there were 85 events of MI or stroke and 37 coronary
revascularizations. Overall, study participants who subsequently
experienced cardiovascular events (cases) had significantly higher
baseline homocysteine levels than those who remained free of reported
cardiovascular disease during follow-up (controls)(14.1 vs 12.4
µmol/L; P=.02). Similar effects were seen in
subgroup analyses of those who did and did not report baseline use of
multivitamin supplements (Table 2).

In age- and smoking status–matched analyses, the risk of any
future cardiovascular event appeared to increase with increasing level
of homocysteine such that the RRs for women in the lowest (referent) to
highest quartiles of homocysteine level at baseline were 1.0, 1.1, 1.1,
and 2.0 (P=.02 for trend). For the end points
of MI or stroke, age- and smoking status–matched risks for individuals
in the lowest (referent) to highest quartiles of homocysteine level
were 1.0, 0.7, 1.6, and 2.2 (P=.004 for trend) (Table 3).

In continuous logistic regression analyses, which evaluated for
evidence of a linear relationship, each increase in homocysteine
concentration of 5 µmol/L was associated with a 20% increase in risk
of any cardiovascular event (P=.03). After
adjustment for baseline differences in other coronary risk factors,
each 5-µmol/L increase in homocysteine was associated with a 24%
increase in risk (P=.05).

To evaluate for evidence of nonlinear effects, we computed RRs across a
series of prespecified cut points defined by the control distribution.
As shown in Table 4, the age- and
smoking status–matched risks of future cardiovascular events for those
with baseline homocysteine levels at or higher than the 50th,
75th, 90th, and 95th percentiles (as defined by the control
distribution) were 1.5 (P=.09), 1.9
(P=.007), 2.0
(P=.03), and 2.6
(P=.02), respectively. Risk estimates were
minimally altered after controlling for baseline differences in
body mass index, exercise frequency, hypertension, hyperlipidemia,
diabetes, and family history of premature MI (Table 4). Controlling
for hormone replacement therapy had no effect on these findings.

Table Graphic Jump LocationTable 4. Crude and Adjusted Relative Risk of Future Cardiovascular Events Among Subjects With Baseline Homocysteine Levels at or Higher Than the 50th, 75th, 90th, and
95th Percentiles of the Control Distribution*

Thirty-four percent of the study participants reported use of
multivitamin supplements at study entry, when blood samples were
obtained. Overall, control women who reported taking multivitamin
supplements had significantly lower mean levels of homocysteine
compared with control women not taking multivitamin supplements (10.9
vs 13.1 µmol/L; P<.001). Nonetheless, additional control
for multivitamin supplement use had little effect on the association of
baseline homocysteine levels with
subsequent risk of cardiovascular disease (Table 3).

To further evaluate this issue, we performed a subgroup analysis
limited to women taking multivitamin supplements at baseline (Table 5). In this subgroup, the
overall distribution of homocysteine level was lower than in the cohort
as a whole, and those with baseline levels of homocysteine at or higher
than the 75th percentile of the reduced control distribution had a 2- to 3-fold increase in risk of any cardiovascular event compared with
those with lower levels (RR, 2.6; 95% CI, 1.2-5.7;
P=.02). Furthermore, the RRs in this subgroup
for any cardiovascular event increased from lowest (referent) to
highest quartiles of baseline homocysteine concentration (RR, 1.0, 0.9,
1.6, and 3.1, respectively; P=.01 for trend).
Similar increases in risk were observed across quartiles of baseline
homocysteine level in the subgroup of women who were not taking
multivitamin supplements at study initiation
(P=.04 for trend) (Table 5).

COMMENT

In this prospective study of apparently healthy
postmenopausal women, those with elevated baseline plasma
concentrations of homocysteine had increased risk of future
cardiovascular events, particularly at the highest levels of
homocysteine. This association appeared to be independent of several
cardiovascular risk factors. Furthermore, baseline multivitamin
supplement use in this cohort was associated with significantly reduced
homocysteine levels. However, in these data, the association between
homocysteine level and cardiovascular risk remained significant in
analyses controlling for baseline multivitamin supplement use, as well
as in subgroup analyses limited to those women who were and were not
taking multivitamin supplements at study entry.

Previous prospective studies of homocysteine level as a
risk factor for coronary heart disease have been inconsistent, with
both positive8,12- 17 and null6,7,9- 12 results
reported. Furthermore, prior studies of homocysteine level in healthy
populations have predominantly been limited to men. Thus, the current
data from postmenopausal women add to the accumulating evidence
regarding homocysteine level as a marker for cardiovascular risk. Our
finding that each 5-µmol/L increase in homocysteine level was
associated with a 20% increase in risk is consistent with
estimates from prior retrospective3 studies and some,
but not all, prospective studies.15 In addition, our data
among apparently healthy women are consistent with subgroup analyses
from the recent ARIC study, in which a positive association was found
in univariate analyses for women but not for men.12 Whether
these observations indicate a chance effect or provide evidence of
effect modification by sex will require future studies.

Our observation that multivitamin supplement use was associated
with reduced homocysteine level is also consistent with prior studies
demonstrating that folic acid intake decreases total plasma
homocysteine level.21,22 In these data, however, a
statistically significant association between homocysteine
concentration and cardiovascular risk was observed in the total cohort
after adjustment for multivitamin supplement use, as well as in
subgroup analyses stratified by multivitamin supplement use.

Potential limitations of these data merit consideration. Our
study measured homocysteine levels only once, at study entry, and thus
are susceptible to regression-dilution bias. Any such bias, however,
would tend to lead to an underestimation of true effects and would
not yield a false-positive result. Also,because
the follow-up period for our study was relatively short (3 years), we
cannot exclude the possibility that homocysteine levels are elevated
because of the presence of subclinical atherosclerosis. This
possibility has been raised in at least 1 prior prospective study of
men in which homocysteine level was found to be a better predictor of
short-term risk than long-term risk.8,9 On the other hand,
because the blood samples in our study were obtained prior to the onset
of first cardiovascular events, we can exclude the possibility that the
elevations observed in our data are a result rather than a cause of
acute vascular occlusion. In addition, we did not ascertain allele
status for the methylenetetrahydrofolate reductase gene in this
population. However, a series of studies has reported that
methylenetetrahydrofolate reductase status alone is a poor indicator of
risk and that assessment of this polymorphism does not substantially
add to the predictive value of homocysteine levels.12,23- 25
Moreover, as in any prospective epidemiological study, the potential
for residual confounding cannot be excluded. In our analysis, however,
we matched on smoking status and age and additionally controlled for
body mass index, hyperlipidemia, exercise frequency, hypertension,
diabetes, hormone replacement therapy, and family history of premature
coronary artery disease. Thus, we believe the magnitude of any residual
confounding is likely to be small.

In conclusion, in this prospective study, women with higher levels of
homocysteine at baseline had higher risks of future cardiovascular
disease. These risks, however, were modest in absolute size and smaller
than those observed in this cohort for other emerging cardiovascular
risk factors, such as high-sensitivity C-reactive
protein.26 It is clear that folate supplementation
reduces homocysteine level,21,22 but whether folic acid
reduces the risk of cardiovascular disease and, if so,
whether such an effect is mediated by lowering homocysteine level
remains a research question that must be tested in randomized
controlled trials. We believe the current totality of evidence is
insufficient on which to base rational clinical decisions for
individual patients or for the general public in terms of any
recommendation to screen for homocysteine level.

Brattstrom L, Wilcken DEL, Ohrvik J, Brudin L. Common
methylenetetrahydrofolate reductase gene mutation leads to
hyperhomocysteinemia but not to vascular disease: the result of a
meta-analysis. Circulation.1998;98:2520-2526.

Table Graphic Jump LocationTable 4. Crude and Adjusted Relative Risk of Future Cardiovascular Events Among Subjects With Baseline Homocysteine Levels at or Higher Than the 50th, 75th, 90th, and
95th Percentiles of the Control Distribution*

Brattstrom L, Wilcken DEL, Ohrvik J, Brudin L. Common
methylenetetrahydrofolate reductase gene mutation leads to
hyperhomocysteinemia but not to vascular disease: the result of a
meta-analysis. Circulation.1998;98:2520-2526.

Letters

The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.
The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with
the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.

This feature is provided as a courtesy. By using it you agree that that you are requesting the material solely for personal, non-commercial use, and that it is subject to the AMA's Terms of Use. The information provided in order to email this article will not be shared, sold, traded, exchanged, or rented. Please refer to The JAMA Network's Privacy Policy for additional information.

Athens and Shibboleth are access management services that provide single sign-on to protected resources. They replace the multiple user names and passwords necessary to access subscription-based content with a single user name and password that can be entered once per session. It operates independently of a user's location or IP address. If your institution uses Athens or Shibboleth authentication, please contact your site administrator to receive your user name and password.